MECHANISMS

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MECHANISMS
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MechanismsStep 1 - Toxicant Delivery

• Absorption

• Presystemic Elimination

• Traverse epithelial barriers and reach blood
  capillaries by diffusing through cells.
• Rate prop to C prop to exposure and dissolution
  and surface area of absorbing surface.
• Lipid-soluble chemicals absorbed more readily
  than H2O-soluble chemicals.

• GIT and lungs are major players (e.g., ethanol,
  morphine), leads to first-pass elimination
  decreases toxicity of chemical before they can
  ever harm the rest of the body.

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• Distribution Towards Target
• Enhanced by

• Distribution Away from Target
• Enhanced by

• Porosity of capillary endothelium due to large
  fenestrae that permit passage of even
  protein-bound xenobiotics.
• Specialized membrane transport (e.g.,
  ATPase-hydrolysis, MPTP.
• Accumulation in cell organelles (e.g., MPTP
  accumulates in the mitochondria of dopamine
  neurons).

• Binding to plasma proteins (e.g., DDT, TCDD bind
  high-MW proteins in plasma, preventing their
  escape from capillaries by diffusion).
• Specialized barriers (e.g., BBB endothelial cells
  lack fenestrae, have tight junctions, oocyte
  surrounded by granulosa cells, spermatogenic
  cells surrounded by Sertoli cells all prevent
  hydrophillic substances from gaining entry).
  However, lipophillic substances can gain entry.
• Distribution to storage sites (e.g., bone,
  kidneys, liver, fat) protect target tissues from
  toxicants

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• Reversible intracellular binding (e.g., by
  binding to the pigment melanin, PAHs accumulate
  in melanin-containing cells also, release of
  melanin-bound toxicants contributes to the
  retinal toxicity assoc. with chlorpromazine and
  chloroquine injury to SN neurons by MPTP, and
  induction of melanoma by polycyclic aromatics).

• Association with intracellular binding proteins
  (e.g., metallothionen binds Cd in the event of
  acute Cd intoxication).
• Export from cells. Intracellular toxicants may
  be transported back into the extracellular space
  (e.g., brain capillary endothelial cells, which
  contain in their luminal membrane an
  ATP-dependent membrane transporter
  (P-glycoprotein) - contributes to the BBB.

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• Reabsorption

• Excretion

• High lipid solubility.
• Many glucuronide conjugates.
• Lipid-soluble compounds are reabsorbed by
  transcellular diffusion.

• Removal of xenobiotics from the blood and their
  return to the external environment
• Renal glomeruli hydrostatically filter small
  molecules (lt 60 kDal) through their pores
  (HOH-soluble and ionized).
• Transporters in hepatocytes and proximal tubular
  cells are specialized for the secretion of highly
  hydrophillic organic acids and bases eliminated
  in bile and urine high pH-dependency

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• Toxification

• Detoxification

• Sometimes, it is the chemical itself is directly
  toxic (e.g., HCN, CO, strong acids and bases,
  nicotine).
• Most often, however, toxification renders
  xenobiotics and sometimes, autocoids (O2 and NO)
  indiscriminantly reactive towards endogenous
  molecules
• Electophiles
• (e.g., chloroform overhead)
• Free Radicals (next 2 slides)
• Nucleophiles - Relatively rare (e.g.,
  dihalomethandes undergo oxidative dehalogenation
  to yield CO).
• Redox-active reactiants
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• Eliminates the ultimate toxicant or prevents its
  formation.
• Detoxification of toxicants with no functional
  groups (e.g., benzene or toluene) undergoes Phase
  I and Phase II reactions - readily excreted as
  hydrophillic organic acids.
• Detoxification of Nucleophiles - Phase II
  reactions prevent peroxidase-catalyzed conversion
  of the nucleophiles to FRs and biotransformation
  of phenols, catechols and hydroquinones to
  electrophiles.
• Detoxification of Electrophiles - Commonly occurs
  with the thiol nucleophile GSH, facilitated by
  GSH-S-transferase.
• Detoxification of FRs. Generation of FRs must be
  undone by various enzymes - See handouts for
  generation of superoxide anion and for its
  elimination by potentially 3 different enzymes.
• Detoxification of protein toxins - many venoms
  contain intramolecular disulfid bonds, which can
  be reduced by an endogenous dithiol protein
  thioredoxin.

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• In summary, the most reactive metabolites are
  electron-deficient molecules and molecular
  fragments such as neutral or cationic free
  radicals.
• Other FRs with an extra electron cause damage by
  yielding the neutral OH after the formation and
  subsequent homolytic cleavage of HOOH.

• When Detoxification Fails
• Toxicants may overwhelm detox processes, leading
  to exhaustion of the detox enzymes, consumption
  of substrates, or depletion of antioxidants such
  as GSH, Asc. acid, and alpha-tocopherol.
• Occasionally, a reactive toxicant inactivates a
  detoxicating enzyme (e.g., ONOO- incapacitates
  Mn-SOD, which would normally counteract ONOO-
  formation.
• Some conjugation reactions can be reversed (e.g.,
  bladder carcinogen 2-naphthylamine is
  hydroxylated and glucuronidated in liver and
  excreted into urine while in bladder, the
  glucuronide is hydrolyzed and the released
  arylhydroxylamine is protonated and dehydrated to
  the reactive electrophile arylnitrenium ion).
• Detoxification can sometimes generate potentially
  harmful by-products, such as GSH thyl radical and
  GSH disulfide, which are produced during the
  detoxification of FRs.

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Step 2 Reaction of the Ultimate Toxicant with
the Target Molecule

• I. Attributes of the target molecules
• Reactivity
• Accessibility
• Critical function
• Reactive metabolites that cannot find appropriate
  endogenous molecules in close proximity to their
  site of formation may diffuse until they
  encounter such reactants.
• Not all chemical targets contribute to harmful
  effects (e.g., CO has deleterious consequences
  when it binds the heme in Hb, but not when it
  binds the heme in cytochrome P-450.
• II. Reaction Types
• 1. Noncovalent binding ionic, H, van der
  Waals interaction of toxicants with receptors,
  ion channels, some enzymes, intracellular
  receptors (e.g., strychnine binds using these
  interactions when binding the GABA receptor in
  the spinal cord TCDD to the Ah receptor
  saxitoxin to Na channels)
• Such forces are also responsible for
  intercalation of chemicals such as acridine
  orange into the DNA double helix

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2. Covelent binding Irreversible. Thus greatly
and permanently alters endogenous molecules.
E.g., elecrophillic reactions with nucleophillic
atoms neutral FRs to DNA. 3. Hydrogen
Abstraction Fenton Reaction (Fig. 3-4) and lipid
peroxidation (Fig. 3-9). 4. Electron
Transfer Oxidation of the Fe in Hb from II to
III produces methemoglobinemia. 5. Enzymatic
Reactions a few toxins act like enzymes in
specific target proteins (e.g., ricin inhibits
protein synthesis by hydrolyzing ribosomes).
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• III. Outcomes Effects of Toxicants on Target
  Molecules
• Dysfunction of Target Molecules some toxicants
  activate endogenous molecules, such as mimicking
  endogenous ligands (e.g., morphine to opiate
  receptors phorbol esters to protein kinase C).
• More commonly, many toxins inhibit function of
  endogenous molecules e.g., atropine, curare, or
  strychnine inhibit Ach receptor-medieated nerve
  transmission.
• Also, interference with template function in DNA
  e.g., covalent binding of aflatoxin 8,9-oxide to
  N7 of G results in base-pairing of the G with A,
  rather, than with C, leading to incorrect codon
  and a point mutation.
• Destruction of Target Molecules Cross-linking
  of endogenous molecules e.g., OH radicals can
  cross-link macromolecules into reactive
  electrophiles, such as protein carbonyls.
• Also, lipid peroxidation (See Fig. 3-9).
• Neoantigen Formation Occasionally, covalent
  binding of xenobiotics or their metabolites to
  macromolecules results in Ab production in some
  individuals e.g., CYP-450 biotransforms
  halothane to an electrophile (trifluoroacetyl
  Cl), which binds as a hapten to various
  microsomal and cell surface proteins in the
  liver, thereby inducing an immune response (Ab
  production).

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Step 3 Cellular Dysfunction and Resultant
Toxicities

• I. Toxicant-Induced Cellular Dysregulation
• A. Dysregulation of Gene Expression
• Occurs at elements that are directly responsible
  for transcription, signal transduction pathways,
  synthesis, storage, or release of extracellular
  signaling molecules.
• B. Dysregulation of Transcription
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• Xenobiotics may interact/promote/interfere with
  promotor regions, interfering with transcription
  factors (TFs).
• e.g., many hormones, vitamins influence gene
  expression by binding to and activating TFs.
• Xenobiotics may mimic the natural compounds
  (e.g., Cd2 may substitute for Zn2 at the
  metal-responsive element-binding TF.
• e.g., estrogen acts as a mitogen normally in
  female reproductive organs but prolonged
  exposure induces tumor formation in these organs.
• e.g, TCDD, Phenobarbital, and pregnenolone
  activate the AhR, thereby inducing CYP1A1

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• C. Dysregulation of Signal Transduction
• Fig. 3-11 in (painstaking) detail NEXT SLIDE.
• Xenobiotics may increase phosphorylation too much
  (e.g., TCDD-liganded AhR binds MAPK may
  contribute to TCDD-induced overexpression of
  kinase activity in liver.
• Abberant phosphorylation or proteins may result
  not only from increased phosphorylation, but also
  from decreased phosphatase activity, such as is
  seen with various chemicals, oxidative stress,
  and UV radiation, which inhibit phosphatase
  activity
• e.g., arsenite, tributyltin, and oxidants, such
  as HOOH, cause phosphorylation of the EGF
  receptor by interfering with the tyr phosphatase
  that would dephosphorylate this receptor.
• e.g., PP2A dephosphorylates MAPK, keeping MAPK
  under control.
• However, some naturally occurring toxins are
  extremely potent inhibitors of PP2A (e.g.,
  blue-green algae poison microsystin-LG and the
  dinoflagellate-derived okadeic acid), which are
  tumor-promotors in experimental animals exposed
  to prolonged low doses.

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• Acute high dose of microsystin-LG ? severe liver
  injury.
• Acute high dose of okadeic acid ? diarrhetic
  shellfish poisoning.
• But this may be due to hyperphosphorylation of
  proteins other than those involved in
  intracellular signaling (e.g., microfillaments)
  e.g., phosphorylation of tau in Alzheimers may
  be caused by too much FRs, oxidant production,
  tyr phosphatase inhibited?

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• D. Dysregulation of Extracellular Signal
  Production
• Hormonal intereference (e.g., herbicide amitrole
  inhibit thyroid hormone production Phenobarbital
  enhance thyroid hormone elimination)
•  
• Both decrease thyroid hormone levels and increase
  the secretion of TSH because of the reduced
  feedback inhibition.
• Another e.g., estrogens (e.g., xenoestrogen
  chlordecone) produce male testicular atrophy by
  means of feedback inhibition of gonadotropin
  secretion ? low sperm count.

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• E. Dysregulation of Ongoing cellular activity
• Note the myriad agents that act on signaling
  systems for neurotransmitters and causing
  dysregulation of momentary activity of
  electrically excitable cells such as neurons and
  myocytes.
• F. Dysregulation of the activity of other Cells
• Many exocrine secretory cells are governed by
  muscarinic Ach receptors, e.g., lacrimation and
  salivation are due to organophosphate insecticide
  poisoning due to stimulation of these receptors.

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• II. Toxic Alteration of Cellular Maintenance
• A. Impairment of Internal Cellular Maintenance
  Mechanisms of Toxic Cell Death
• ATP depletion
• Sustained rise in intracellular Ca2
• Overproduction of ROS and RNS
• Depletion of ATP. Recall Oxidative
  Phosphorylation mitochondrial function of ATP
  production, delivery of H in the form of NADH,
  formation of the H gradient, O2 delivery to the
  terminal electron-transport complex and the
  reduction of O2 to H2O.
• Sustained rise in intracellular Ca2. Fig. 3-14.
  Influenced by 3 Mechanisms
• High cytoplasmic Ca2 levels cause increased
  mitochondrial Ca2 uptake by the Ca2 uniporter,
  which uses the mitochondrial internal negative
  membrane potential as the driving force. The
  membrane potential, therefore, dissipates and ATP
  production ceases.
• ATP synthesis is impaired by Ca2-induced
  oxidative injury to the inner mitochondrial
  membrane
• (3) ATP consumption is increased due to the
  Ca2-ATPase working overtime to eliminate the
  excess Ca2.
• Overproduction of ROS and RNS (recall Fig. 3-3
  and Fig. 3-4).

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Interplay Among Primary Metabolic Cell Disorders
Spells Disaster For The Cell

• Mito. Permeability Transfer (MPT) and the worst
  possible outcome Necrosis.
• MPT ? permeability of the mitochondrial inner
  membrane
• ? Mitochondrial Ca2 uptake
• ? ??m
• ? ROS, RNS
• ? ATP
• ? Pi, FFAs

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• Proteinaceous pore opens up and spans both
  mitochondrial membranes (pore size lt 1.5 kDal).
• Causes free influx of Ca2, H2O, dissipation of
  the ??m, osmotic swelling, ? ATP synthesis,
  lysis.
• Also, depolarization of inner mito. membranes
  ?ATP synthesis to operate in reverse, as in
  ATPase, hydrolyzing ATP.
• Also, glycolysis may become compromised by
  insufficient ATP to supply ATP-requiring enzymes
  (e.g., PFK, hexokinase).
• Then, oxidative and hydrolytic degredation of
  macromolecules and membranes and degredation of
  intracellular milieu ? cell lysis (necrosis).

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Alternative Outcome of MPT Apoptosis

• Necrosis Cell

• Apoptotic Cell

• Swells
• Lysis
• Chaotic, random sequence of events

• Shrinks
• Cyto and nuclear materials condense, break off
  into membrane-bound fragments (apoptotic bodies)
  that are phagocytosed.
• Membranes bleb.
• Ordered, cascade-like activation of catabolic
  processes that disassemble the cell.

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ATP Availability Determine the Form of Cell Death

• Common features of Necrosis and Apoptosis
• Many xenobiotics (e.g., acetominophen, ocratoxin)
  cause both.
• A. Apoptosis induced at low exposure levels and
  early after high exposure levels.
• B. Necrosis later at higher exposure levels.
• Similar metabolic disturbances, e.g., MPT.
• Blockers of necrosis (e.g., Bcl2 over-expression
  will block apoptosis.

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ATP Availability is Critical in Determining the
form of Cell Death
MPT
In few mito and cytochrome c are removed
by lysosomal autophagy
In all mito. ATP Severely depleted. Cannot
execute apoptotic program (requires
ATP). Cytolysis occurs before caspases
are activated.
In a few more mito autophagic mech are
overwelmed released cyt c initiates caspase and
activation and apoptosis.
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Step 4 Repair or Disrepair

• MOLECULAR REPAIR
• Repair of proteins
• Some Methods
• Repair of Oxidized Hb (Met Hb) e- transfer from
  cyt. b5 from cyt b5 reductase (-NAHPH-dependent).
• Chaperones e.g., heat-shock proteins refold
  altered proteins.
• Proteosomes proteolytically degrade mutated
  protein.

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• Repair of Lipids.
• NAPDH-dependent repair of peroxidized lipids
  using GSH peroxidase and reductase.
• Repair of DNA.
• Despite high reactivity with electrophiles and
  FRs, nuclear DNA is quite stable, partly because
  its packaged into chromatin, highly folded, and
  has several repair mechanisms. Mitochondrial
  DNA, however, is more prone to damage because it
  lacks histones and efficient repair mechanisms.

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Some DNA-Damaging AgentsFour Major Categories

• Direct-acting carcinogens intrinsically
  reactive and dont require metabolic activation
  by cellular enzymes to covalently interact with
  DNA e.g., N-methyl-N-nitrosourea,
  alkylsulfonates (methyl methanesulfonate),
  lactones (beta-propriolactone), and N and S
  mustards.
•  Indirect-acting carcinogens require metabolic
  activation by cellular enzymes to form the
  ultimate carcinogenic species that covalently
  binds to DNA e.g., dimethylnitrosamine,
  benzoapyrene, 7,12-dimethylbenzaanthracene,
  aflatoxin B1, and 2-acetylaminofluorene.

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Some DNA-Damaging AgentsFour Major Categories
(contd)

• Radiation and oxidative damage can occur either
  indirectly or directly ionizing radiation to
  produce ds breaks or ionization of water to
  produce ROS/RNS that damage DNA bases. UV
  radiation also.
• 4. Inorganic agents (e.g., As, Cr, Ni), but many
  mechanisms are unknown but 3 general types of
  genetic alterations can result

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Some DNA-Damaging AgentsFour Major Categories
(contd)

1. Gene mutations point- (bp-substitutions) and
   frame-shift mutations.
2. Chromosome aberrations (gross rearrangements,
   deletions, duplications, inversions, and
   translocations).
3. Aneuploidy, polyploidy.

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• Direct Repair DNA photolyase
• Excision Repair Base/nucleotide excision,
  ligase, polymerase, PARP.
• Recombinatorial Repair
• --sister chromatid exchange.
• --Occurs when the excision of a bulky adduct or
  an intrastrand pyrimidine dimer fails to occur
  before DNA replication begins.
• --Cross-over and recombination.

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• CELLULAR REPAIR
• Nonneural tissue divide to replace lost cells.
• Neurons dont multiply.
• PNS Macrophages and Schwann Cells
• Phagocytosis
• and
• Growth factors and cytokines
• facilitate transdifferentiation from
• myelination mode to growth-
• supporting mode.
• CNS Lost neurons are compensated by
• neighboring neurons.

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• TISSUE REPAIR
• Tissue is composed of t components
• cells and ECM. 2 Processes
• Apoptosis Deleting damaged cells.
• Proliferation Regeneration of (damaged)
  tissue.
• Replacement of lost cells by
  mitosis.

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• Replacement of the ECM.
• In liver stellate cells and adipocytes
• (spaces of Disse)
• ECM proteins
• glycosaminoglycans
• glycoprotein
• proteoglycan glucoconjugates

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• When Repair Fails
• Repair mechanisms are not absolute can overlook
  various lesions.
• Repair can contribute to toxicity.
• e.g., after chronic tissue injury when the
  repair process goes astray and leads to
  uncontrolled proliferation, rather than tissue
  remodeling. Such proliferation of cells may
  yield neoplasms, whereas overproduction of ECM
  results in fibrosis.

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• Toxicity Resulting from Disrepair
• Tissue Necrosis.
• e.g, lipid peroxidation can be repaired unless
  a-tocopherol is depleted ? cell injury progresses
  toward cell necrosis if molecular repair
  mechanisms are insufficient or the molecular
  damage is not readily reversible.
• -- Fibrosis.
• Pathological condition characterized by
  excessive deposition of abnormally composed ECM
  or too much ECM. E.g., chronic alcohol leading
  to hepatic fibrosis and cirrhosis.

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• --- Carcinogenesis
• Chemical carcinogenesis involves insufficient
  function of various repair mechanisms including
• (1) Failure of DNA repair.
• (2) Failure of Apoptosis.
• (3) Failure to terminate cell proliferation.

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• Failure of DNA repair Mutation is the
  initiating event in carcinogenesis.
• DNA damage adduct formation
• oxidative changes
• strand breakage
• Failure to repair lesion ? heritable alteration
  (mutation) in the daughter strands during
  replication. Mutations can reprogram cells for
  multiplication. Enhanced cell divisions ?
• likelihood of mutations.

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• Proto-oncogenes.
• Highly conserved genes encoding proteins that
  stimulate the progression of cells through the
  cell cycle.
• Products are growth factors, GF receptors,
  intracellular signaling transducers, such as G
  proteins, protein kinases, and nuclear
  transcription factors.
• Genetic carcinogens mutate proto-oncogenes ?
  Oncogene.
• Constitutive sustained activation of the
  promotor region of the proto-oncogenem e.g.,
  TCDD or benzo(a)pyrene can activate the Ah
  receptor ? binds the promotor on Ras ?
  transactivation in perpetuity.

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• Tumour-Suppressor Genes
• p53 Guardian of the genome because it
  eliminates cancer-prone cells from the
  replicative pool, counteracting neoplasmic
  transformation.
• Mutations in p53 gene found in 50 of human
  tumors and in a variety of induced cancers.
• Cells with no p53 are 106 times more likely to
  permit DNA amplification then are cells with a
  normal level of p53 suppressor protein.

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• Cooperation of Proto-oncogenes and Tumor
  Suppressor Genes in Carcinogenesis
• The accumulation of genetic damage is due to
  mutant proto-oncogenes and mutant tumor
  suppressor genes.
• ? This is the main driving force in the
  transformation of normal cells with controlled
  proliferative activity (normal mitosis) to
  malignant cells with uncontrolled proliferative
  activity (cancer cells, metastatic).
• Because the normal number of cells in a tissue is
  regulated by a balance between mitosis and
  apoptosis, uncontrolled proliferation results
  from a disturbance of this balance.

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• (2) Failure of Apoptosis Promotion of mutation
  and clonal growth.
• Apoptosis eliminates cells with damaged DNA,
  preventing mutation, which initiates
  carcinogenesis.
• Inhibiting apoptosis facilitates mutation and
  clonal expansion of pre-neoplastic cells
• e.g., phenobarbital.

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• (3) Failure to Terminate Replication.
• A. Enhanced mitotic activity ? P(mutations).
• G1 phase of cell cycle is shortened ? ? time
  used for DNA repair ?? P(mutations).
• B. During increased proliferation,
  proto-oncogenes are over-expressed, which may
  cooperate with oncogene proteins to facilitate
  neoplastic transformation of cells. ? ? time for
  DNA methylation at C5 of C residues ? enhances
  gene expression and may result in over-expression
  of proto-oncogenes and oncogenes.

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• C. Cell-cell communication through gap
  junctions and intercellular adhesion through
  cadherins are temporarily disrupted during
  proliferation.
• Lack of these junctions contributes to the
  invasiveness of tumor cells.
• Several tumor promotors (phenobarbitol, phorbol
  esters) ? gap junctions and intercellular
  communication.